The present invention relates to a method for maintaining the quality of aqueous injection preparation of thrombomodulin in a non-frozen or non-freeze-dried liquid form over its storage/transportation and to an aqueous injection preparation of thrombomodulin stable over its storage/transportation.
Thrombomodulin (in the following, abbreviated sometimes as TM) is a substance having a function of coupling specifically with thrombin and accelerating the activation of protein C by thrombin remarkably. Protein C consists of a vitamin K dependent protein which plays an important role in the coagulation fibrinolysis system and is activated by the action of thrombin into activated protein C. It has been known that activated protein C will inactivate the activated coagulation factors V and VIII in the blood coagulation system of living body and that it participates in the production of plasminogen activator which reveals a thrombolytic function (See Koji Suzuki, xe2x80x9cIgaku-no Ayumi (Progress in Medical Science)xe2x80x9d, Vol. 125, 901 (1983))
Thus, it has been recognized that thrombomodulin accelerates activation of protein C by thrombin to produce a large amount of activated protein C exhibiting inhibitive action to blood coagulation and thrombolytic function and is useful as an anticoaglant and as a thrombolytic agent. It has heretofore been expected to use thrombomodulin for therapy and prophylaxis of, such as acute coronary syndrome (ACS), for example, myocardiac infarction and unstable angina, for reconstruction of coronary circulation; for therapies and prophylaxes of thromboses, such as acute and chronic cerebral thromboses and acute and chronic arterial and venous peripheral thromboses; peripheral blood vessel obstruction, such as Buerger disease and Raynaud disease; obstructive arteriosclerosis; blood vessel inflammation, such as systemic lumpus erythematodes (SLE), Becet disease and Kawasaki disease; and for therapies and prophylaxes of functional disorders pursuant to a cardiac surgery, complications pusuant to an organ transplantation, disease of intravascular coagulation (DIC), angina pectoris, transient cerebral ischemic attack, gestational toxicosis, diabetes, liver veno-occlusive diseases (VOD), such as venoocclusive diseases following fulminant hepatitis or after bone marrow transplantation, and deep venous thrombosis (DVT).
In the past, thrombomodulin was confirmed and isolated as a glycoprotein appearing on the endothelial cells of blood vessel of animals, including human. Our group of inventors of the present invention had succeeded to clone it for the first time. Thus, the inventors had performed cloning of genes of a precursor of a human thrombomodulin having a signal peptide from the human lung cDNA library using a technique of genetic engineering, whereby the entire gene sequence of the thrombomodulin was analyzed and its amino acid sequence having 575 residues including a signal peptide of 18 amino acid residues was made clear (See Japanese Patent Kokai Sho 64-6219 A). It has been known that a mature thrombomodulin with the signal peptide being cut is composed of five domains, namely, the NH2-terminal domain (1st to 226th amino acid residue, in the order from the NH2-terminus), the domain having 6 EGF-like structures (227th to 462nd residue), the O-glycosylation site-rich domain (463rd to 498th residue), the transmembrane domain (499th to 521st residue) and the cytoplasmic domain, enumerated respectively in the sequential order from the NH2-terminus, wherein the segment exhibiting the activity corresponding to that of the full length thrombomodulin, namely, the least active unit thereof, is constituted of the segemnt consisting of the fourth, fifth and sixth EGF-like structures in the domain having 6 EGF-like structures as enumerated in the order from the NH2-terminus (See M. Zushi et al, J. Biol. Chem., 246, 10351-10353 (1989)).
It has been confirmed that at least such a thrombomodulin which is prepared so as to eliminate the transmembrane domain has a nature of being clearly dissolved in water even without using any surfactant (in the following, referred to sometimes as xe2x80x9csoluble thrombomodulinxe2x80x9d) and, for example, a thrombomodulin composed of only the three domains, i.e. the NH2-terminal domain, the domain having 6 EGF-like structures and the O-glycosylation site-rich domain, namely, composed of the amino acid sequence from the 19th to 516th amino acid residues of the sequence listing SEQ ID NO:1, can be obtained by applying a gene recombination technology and that the so-obtained recombinant thrombomodulin has an activity which is the same as that of natural thrombomodulin (Japanese Patent Kokai Sho 64-6219 A).
By the way, as recognized in many cases, genes may suffer from natural and artificial mutations due to inspection work upon, for example, their isolation etc., without exception for human in which also a polymorphic mutation had been discovered, wherein two mutants of the precursor of human thrombomodulin constituted of the amino acid sequence composed of the above-mentioned 575 amino acid residues were confirmed, in which the amino acid residue at the 473rd site consists of Val, for the one, and in which this amino acid residue consists of Ala, for the other. This corresponds, in the base sequence coding such amino acids, to mutations of T and C of the 1418th site, respectively (Wen et al, Biochemistry, 26, 4350-4357 (1987)). They do reveal, however, no difference in the activity and in the physical properties from each other and can be regarded as being substantially identical. Therefore, the above-mentioned human thrombomodulin constituted of the amino acid sequence of the sequence listing SEQ ID NO:1 is regarded as one of polymorphs of the peptide of thrombomodulin composed of the amino acid sequence of sequence listing SEQ ID NO: 2 and both should be judged as substantially identical with each other.
On the other hand, thrombomodulin preparations have currently been distributed steadily for practical use in a form of freeze-dried medicinal pharmaceutical. Meanwhile, it had been discovered that, in the process of freeze-drying of a thrombomodulin-containing aqueous solution, a part of thrombomodulin is converted, though in a minute amount, into a polymeric matter due to a denaturization to form a polymer in which several molecules of thrombomodulin are held in association. For resolving this problem, the inventors made sound researches, whereby they reached the discovery that denaturization of thrombomodulin upon freeze-drying thereof could be prevented, of which invention was previously applied for a patent (Japanese Patent Kokai Hei 6-321805 A).
Despite of the above circumstances, requests had been raised for providing a novel preparation in a non-frozen or non-freeze-dried form which can be used simply and easily and can be produced at a lower production cost.
The inventors of the present invention had investigated the possibility of realizing an aqueous injection preparation of thrombomodulin which can afford to avoid incorporation of freeze-drying process and to eliminate necessity of dissolution procedure upon practical use, under an attempt of developing a new preparation other than the freeze-dried preparation. While it is requested at first for an aqueous injection preparation that its residual potency after storage over a long period of time at 5xc2x0 C. to room temperature should not be decreased to a considerable extent (i.e. a long term stability), investigations have shown that it is not easy to attain such a long term stability. To our surprise, it was further found that an aqueous injection preparation may in some cases become turbid by shaking it. It is expected enough that a liquid preparation maybe subjected to a shaking motion, though at different intensity levels, on its distribution, such as transportation. Such a problem is not encountered for conventional freeze-dried powdery preparation. Thus, a new and unexpected problem was recognized by us for a stability against shaking of aqueous injection preparation. Presence of any insoluble matter in an injection preparation may cause occasionally a fatal accident in patients, in particular, having disorders in the circulatory organs and, thus, the problem in the stability against shaking for obviating occurence of turbidity upon shaking of aqueous injection preparation mentioned above is a very significant obstruction to be overcome.
In summary, it has heretofore been difficult to prepare an aqueous injection preparation which can permit to store/transport in a liquid form as such over a long period of time, since any injection preparation which is satisfactory with respect to the long term stability and to the stability against shaking can never be obtained by simply dissolving thrombomodulin in water, in despite of the circumstances that an aqueous injection preparation has advantageous features that it can conveniently be administered to patient without necessitating any procedure of dissolving in the water for injection upon practical use and that it can be produced economically and more easily due to elimination of the procedure of freeze-drying, as compared with the conventional freeze-dried preparation.
The inventors had conducted sound researches for obviating the above described problems and found that all these problems were able to be solved by keeping a certain condition, which has led to the completion of the present invention.
Thus, the present invention provides for a method for maintaining the quality of aqueous injection preparation of thrombomodulin in a non-frozen or non-freeze-dried liquid form over its storage and transportation, characterized in that the aqueous injection preparation of thrombomodulin is prepared as an aqueous solution having a pH value in the range from 5 to 7.0 and containing (a) soluble thrombomodulin in an effective amount and a buffer component revealing a buffering action in a pH rang between 5 and 7.0, wherein the aqueous solution of thrombomodulin has either the following characteristic feature a) or b), namely,
a) that it contains further a surfactant and is filled aseptically in a container or
b) that it consists of a prefilled syringe preparation filled aseptically in a syringe container so as to exclude any substantial gas space therein.
Therefore, the first embodiment form of the method for maintaing the quality of aqueous injection preparation according to the present invention consists in that the aqueous injection preparation of thrombomodulin, which is characterised in that it is prepared as an aqueous solution having a pH value in the range from 5 to 7.0 and containing a soluble thrombomodulin in an effective amount, a buffer component revealing a buffering action in a pH range between 5 and 7.0 and a surfactant and is filled in a container aseptically, is stored/transported in a liquid form over a long period of time.
The second embodiment form of the method for maintaing the quality of aqueous injection preparation according to the present invention consists in that the aqueous injection preparation of thrombomodulin, which is characterised in that it is prepared as an aqueous solution having a pH value in the range from 5 to 7.0 and containing a soluble thrombomodulin in an effective amount and a buffer component revealing a buffering action in a pH range between 5 and 7.0 and consists of a prefilled syringe preparation filled aseptically in a syringe container so as to exclude any substantial gas space therein, is stored/transported in a liquid form over a long period of time.
The third favorable embodiment form of the method for maintaing the quality of aqueous injection preparation according to the present invention consists in that the aqueous injection preparation of thrombomodulin, which is characterised in that it is prepared as an aqueous solution having a pH value in the range from 5 to 7.0 and containing a soluble thrombomodulin in an effective amount, a buffer component revealing a buffering action in a pH range between 5 and 7.0 and a surfactant and consists of a prefilled syringe preparation filled aseptically in a syringe container so as to exclude any substantial gas space therein, is stored/transported in a liquid form over a long period of time.
The present invention also provides for a method for realizing a long term stability and a stability against shaking of an aqueous injection preparation of thrombomodulin in a non-frozen or non-freeze-dried liquid form, characterized in that the aqueous injection preparation of thrombomodulin is prepared as an aqueous solution having a pH value in the range from 5 to 7.0 and containing a soluble thrombomodulin in an effective amount and a buffer component revealing a buffering action in a pH range between 5 and 7.0, wherein the aqueous solution of thrombomodulin has either the following characteristic feature a) or b), namely,
a) that it contains further a surfactant and is filled aseptically in a container or
b) that it consists of a prefilled syringe preparation filled aseptically in a syringe vessel so as to exclude any substantial residual gas space therein.
The aqueous injection preparation of thrombomodulin to be used in the method according to the present invention in a non-frozen or non-freeze-dried liquid form, prepared as an aqueous solution having a pH value in the range from 5 to 7.0 and containing a soluble thrombomodulin in an effective amount and a buffer component revealing a buffering action in a pH range between 5 and 7.0, is superior in the stability for long term storage and in the stability against shaking and suitable for storing/transporting over a long period of time and is characterized in that the aqueous solution of thrombomodulin has a pH value in the range from 5 to 7.0 and has either the following characteristic feature a) or b), namely,
a) that it contains further a surfactant and is filled aseptically in a container or
b) that it consists of a prefilled syringe preparation filled aseptically in a syringe vessel so as to exclude any substantial residual gas space therein.
Thus, the first aspect of the aqueous injection preparation of thrombomodulin according to the present invention consists in an aqueous injection preparation of thrombomodulin in a non-frozen or non-freeze-dried liquid form, superior in the stability for long term storage and in the stability against shaking and suitable for storing/transporting over a long period of time, characterized in that the aqueous injection preparation of thrombomodulin has a pH value in the range from 5 to 7.0, contains a soluble thrombomodulin in an effective amount, a buffer component revealing a buffering action in a pH range between 5 and 7.0 and a surfactant and is filled in a container aseptically.
The second aspect of the aqueous injection preparation of thrombomodulin according to the present invention consists in an aqueous injection preparation of thrombomodulin in a non-frozen or non-freeze-dried liquid form, superior in the stability for long term storage and in the stability against shaking and suitable for storing/transporting over a long period of time, characterized in that the aqueous injection preparation of thrombomodulin is a prefilled syringe preparation which has a pH value in the range from 5 to 7.0, contains a soluble thrombomodulin in an effective amount and a buffer component revealing a buffering action in a pH range between 5 and 7.0 and which is filled in a syringe container aseptically so as to exclude any substantial gas space therein.
The third aspect of the aqueous injection preparation of thrombomodulin according to the present invention consists in an aqueous injection preparation of thrombomodulin in a non-frozen or non-freeze-dried liquid form, superior in the stability for long term storage and in the stability against shaking and suitable for storing/transporting over a long period of time, characterized in that the aqueous injection preparation of thrombomodulin is a prefilled syringe preparation which has a pH value in the range from 5 to 7.0, contains a soluble thrombomodulin in an effective amount, a buffer component revealing a buffering action in a pH range between 5 and 7.0 and a surfactant and which is filled in a syringe container aseptically so as to exclude any substantial gas space therein.
The term xe2x80x9cthrombomodulinxe2x80x9d as used in the specification of this patent application refers, without any special restriction, to every substance having a function of being bound to thrombin to bring about acceleration of the activation of protein C by the thrombin. In the context of this specification, the term xe2x80x9csoluble thrombomodulinxe2x80x9d refers to a substance which exhibits an activity as the above-mentioned xe2x80x9cthrombomodulinxe2x80x9d and is soluble in water easily even in the absence of surfactant and which may favorably exhibit a solubility of, for example, at least 1 mg/ml, preferably at least 3 mg/ml, especially preferably at least 6 mg/ml, in the water for injection. As favorable examples of the soluble thrombomodulin, peptides having a molecular weight, as determined by SDS-polyacryamide gel electrophoresis in non-reduced state, of 66,000xc2x110,000, exhibiting a function for accelerating the activation of protein C by thrombin and soluble in the water for injection at least at a concentration of 6 mg/ml may be enumerated. As other favorable soluble thrombomodulins, those peptides which are constituted of amino acid sequences including the amino acid sequence composed of the amino acid residues from the 19th site to the 29th site in the sequence listing SEQ ID NO: 1, exhibiting a function for accelerating the activation of protein C by thrombin and soluble in water are exemplified. Further examples of favorable soluble thrombomodulins include those consisting of the following i) and ii), namely,
i) thrombomodulins which are constituted of an amino acid sequence composed of the 19th site to the 516th site amino acid residues in the sequence listing SEQ ID NO: 1 and
ii) thrombomodulins which comprise an amino acid sequence composed of those amino acids in which one or more amino acid residues in the amino acid sequence given above are replaced or removed or one or more amino acid residues are added thereto,
and exhibiting a function for accelerating the activation of protein C by thrombin.
As the soluble thrombomodulin, there may further be enumerated peptides constituted of amino acid sequences including at least the 4th, 5th and 6th structures, as enumeratd in the sequential order from the NH2-terminus, which are regarded as constituting the smallest active unit of thrombomodulin, in the domain having 6 EGF-like structures (for example, those of Nos. 367 to 480 sites in the sequence listing SEQ ID NO: 1 and the sequence listing SEQ ID NO: 2 for human thrombomodulin). Especially preferred soluble thrombomodulins among them are those peptides which can be produced by transformed cells obtained by transfecting the DNA segment coding the amino acid sequence of the sequence listing SEQ ID NO: 1 or of the sequence listing SEQ ID NO: 2 to a host cell using a vector. As one preferred example of the peptides obtainable from the transformed cells, namely, soluble thrombomodulins, there may be enumerated the peptides constituted of the amino acid sequences composed of the Nos. 19 to 516 sites of the sequence listing SEQ ID NO: 1 and of the sequence listing SEQ ID NO: 2. Others may include, in accordance with each specific host cell, those in which the signal peptide remains as such and which are constituted of the amino acid sequences from the No. 17 to the No. 516 site of the sequence listing SEQ ID NO: 1 and of the sequence listing SEQ ID NO: 2 and mixtures of them. Of course these peptides have quite high solubilities in water which is sufficient for satisfying the prescribed solubility explained above. It is enough for these peptides only that they have the above-mentioned amino acid sequence and no restriction is made therefor, wherein it is no matter whether they have glycosyl chains or not. It is even possible to utilize soluble peptides which are obtainable from human urine and the like. While the kind, the site of addition and the degree of addition of the glycosyl chain in the peptide may be different in accordance with each specific host cell, use of such peptide is not restricted thereby. The peptides composed of the amino acid residues from the No. 367 site to the No. 480 site of the sequence listing SEQ ID NO: 1 and of the sequence listing SEQ ID NO: 2 themselves have a high stability against shaking and may, in any case, result in a favorable aqueous preparation, nevertheless, it is to be understood that it is necessary to increase the stability against shaking by having resort to the technique according to the present invention, for the soluble thrombomodulins having the above-mentioned molecular weight, namely, 66,000xc2x1 10,000 in non-reduced state, for example, the soluble thrombomodulin constituted of the amino acid sequence of the residues from No. 19 site to No. 516 site of the sequence listing SEQ ID NO: 1, the soluble thrombomodulin constituted of the amino acid sequence of the residues from No. 19 site to No. 516 site of the sequence listing SEQ ID NO: 2, the soluble thrombomodulin obtained by transfecting a DNA segment coding the amino acid sequence described in the sequence listing SEQ ID NO: 1 to a host cell and the soluble thrombomodulin obtained by transfecting a DNA segment coding the amino acid sequence described in the sequence listing SEQ ID NO: 2 to a host cell.
For the host cell, there may be enumerated cells of, for example, Chinese hamster ovary (CHO), COS-1, COS-7, VERO (ATCC CCL-81), BHK, canin-originated MDCK and hamster AV-12-664, as well as human-originated cells of, such as HeLa, WI38 and human 293. As the CHO cell, DHFRxe2x88x92 CHO cell is more preferred. In the course of gene manipulation, various microorganisms, such as Escherichia coli and the like, are employed frequently, wherein it is favorable to choose a host-vector system adapted for each purpose and, thus, an adequate vector system should also be selected for each of the above host cells.
The gene of each thrombomodulin to be incorporated in the genetic recombination technique has been cloned and production examples using the genetic recombination technique for thrombomodulin have been disclosed, in addition to the technique of purification for obtaining a purified product (See Japanese Patent Kokais Sho 64-6219 A, Hei 2-255699 A, Hei 5-213998 A, Hei 5-310787 A and Hei 7-155176 A as well as J. Biol. Chem., 264, 10351-10353 (1989)). Therefore, the thrombomodulin to be used according to the present invention can be produced using the techniques described in the above-cited literatures or by means of a technique corresponding to them. For example, there is disclosed in Japanese Patent Kokai Sho 64-6219 A Escherichia coli K-12 strain DH5 (with ATCC Deposition No. 67283) having the plasmid psV2TMJ2 containing the DNA coding the entire length of a thrombomodulin, while the applicant of the present invention made again a deposition of the same strain (Escherichia coli DH5/psV2TMJ2) at the Institute of Life Engineering of Agency of Industrial Science and Technology of the Ministry of International Trade and Industry at No. 1-3, Higashi 1 Chome, Tsukuba City, Ibaragi Prefecture, Japan, on Jun. 19th, 1996. The Receipt No. was FERM BP-5570. Using, as the starting material, this DNA which codes the entire length of thrombomodulin, the soluble thrombomodulin to be used according to the present invention can be obtained by a known gene manipulation technique.
While the soluble thrombomodulin to be used according to the present invention may be produced by a known technique or a technique corresponding thereto, it is possible therefor to refer to the literatures of, for example, Yamamoto et al (Japanese Patent Kokai Sho 64-6219 A, Cf. Examples) and Japanese Patent Kokai Hei 5-213998 A, given above. Thus, it is possible to obtain the DNA coding the amino acid sequence of the sequence listing SEQ ID NO: 1, by processing the gene of a human thrombomodulin by a gene manipulation technique and, if necessary, to modify it. Such a modification may include a site-specific mutation of the codon that codes the amino acid at the 473th site of the sequence listing SEQ ID NO: 1 (especially the base at the 1418th site) for obtaining the DNA which codes the amino acid sequence of the sequence listing SEQ ID NO: 2, in accordance with the method described in Method in Enzymology, 100, 468 (1983), Academic Press. For example, using a DNA fragment containing the base sequence of the sequence listing SEQ ID NO: 3 and a synthetic DNA for mutation containing the base sequence shown in the sequence listing SEQ ID NO: 5, the above-mentioned site-specific mutation is performed to obtain the DNA which codes the amino acid sequence of the sequence listing SEQ ID NO: 2. The so-obtained DNA can then be integrated into, for example, Chinese hamster ovarian cells to obtain corresponding transformed cells, among which cells chosen pertinently are cultured and, from the culture mixture thereof, the soluble thrombomodulin purified by a known technique is obtained. It is favorable, as mentioned previously, that the DNA which codes the amino acid sequence of the sequence listing SEQ ID NO: 1 is transfected to the host cell. The technique for producing the soluble thrombomodulin to be used according to the present invention should not be restricted only to the above-described procedures but many alternatives may be possible including, for example, by recourse to a technique in which the thrombomodulin or soluble thrombomodulin is extracted from a tissue, from a tissue culture mixture, from human urine or so on, with subsequent purification and, if necessary, with further treatment by a proteolytic enzyme.
The isolation and purification of the soluble thrombomodulin from the supernatant or from the culture mixture from the above technique can be performed in a known technique (See, for example, xe2x80x9cFundamental Experimental Methods with Proteins, Enzymesxe2x80x9d, edited by Buichi Horlo). Use of ion-exchange chromatography based on the interaction between the thrombomodulin and a chromatography stationary phase carrier on which a functional group with ionic charge reverse to that of the thrombomodulin is fixed is also preferable. Also an affinity chromatography based on the specific affinity to the thrombomodulin may favorably be exemplified. As a favorable example of adsorbent to be used, there may be enumerated antibodies of thrombomodulin and of thrombin, which may constitute a ligand of the thrombomodulin. For these antibodies, those of thrombomodulin capable of recognizing an adequate function or an adequate epitope can be utilized, examples of which include those disclosed in, for example, Japanese Patent Publication Hei 5-42920 B and Japanese Patent Kokais Sho 64-45398 A and Hei 6-205692 A. Further, a gel filtration chromatography and an ultrafiltration technique realized with respect to the molecular size of the thrombomodulin. Furthermore, there may be employed a hydrophobic chromatography based on a hydrophobic interaction between the carrier of the stationary phase on which a hydrophobic group is bound and the hydrophobic site of the thrombomodulin. The above-mentioned techniques may be employed in an adequate combination. The degree of purification of the soluble thrombomodulin may adequately be selected in accordance with each specific application, while it is favorable to purify it up to such a degree that a single band will be obtained by, for example, electro-phoresis, preferably, SDS-PAGE, or a single peak will be obtained by, for example, gel permeation HPLC or reversed phase HPLC.
Concretely exemplifying the procedures of purification, a technique in which the purification is proceeded taking a thrombomodulin activity into account as a parameter may be employed, wherein, for example, the supernatant or the culture mixture is treated by Q-Sepharose-FF of an ion-exchange column to collect fractions exhibiting an activity of thrombomodulin, which are then subjected to the proper purification by diisopropylphosphoryl thrombin agarose (DIP-TB), followed by collection of fractions exhibiting higher activity of thrombomodulin, which are then subjected to gel permeation chromatography to realize collection of thrombomodulin-active fractions, in order to obtain a pure soluble thrombomodulin product (See Gomi et al, Blood, 75, 1396-1399 (1990)). As the parameter for the activity of thrombomodulin, there may be utilize, for example, the activity of thrombomodulin to accelerate the activation of protein C. As another purification technique, the following method may also be exemplified:
The thrombomodulin is subjected to an ion-exchange chromatography using an adequately selected ion-exchange resin exhibiting a better adsorbing performance for the thrombomodulin. An especially preferred method utilizes Q-Sepharose-FF equlibrated with 0.02 M Tris-HCl buffer solution (pH=7.4) containing 0.18 M NaCl. The retained thrombomodulin can be eluted out after being washed adequately, using, for example, 0.02 M Tris-HCl buffer solution (pH=7.4) containing 0.3 M NaCl, to obtain a crude product of thrombomodulin.
Thereafter, this product may be subjected to a purification step by, for example, an affinity chromatography using a resin on which a substance having a specific affinity to thrombomodulin is bound. As preferable examples, a DIP-thrombin-agarose column and a column with an anti-thrombomodulin monoclonal antibody may be enumerated. The DIP-thrombin-agarose column can be preliminarily treated by being equilibrated with, for example, a 20 mM Tris-HCl buffer solution (pH=7.4) containing 100 mM NaCl and 0.5 mM calcium chloride, whereupon the column is charged with the above-mentioned crude product, followed by an adequate washing with subsequent elution with, for example, a 0.20 mM Tris-HCl buffer solution (pH=7.4) containing 1.0 M NaCl and 0.5 mM calcium chloride, to obtain a purified product of the soluble thrombomodulin. In the case of the anti-thrombomodulin monoclonal antibody column, the column is filled with Sepharose 4B (of the firm Pharmacia) which has been treated in such a manner that the Sepharose particles in a state activated preliminarily by CNBr are brought into contact with a 0.1 M NaHCO, buffer solution (pH=8.3) containing 0.5 M NaCl and an anti-thrombomodulin monoclonal antibody dissolved therein so as to cause the anti-thrombomodulin monoclonal antibody to be bound onto the Sepharose particles. Then, the column is equilibrated beforehand with, for example, a 20 mM phosphate buffer solution (pH=7.3) containing 1.0 M NaCl, followed by washing adequately, before the retained thrombomodulin is eluted out with, for example, a 100 mM glycine-HCl buffer solution (pH=3.0) containing 0.3 M NaCl.
The so-obtained solution of the purified soluble thrombomodulin is then charged in a column filled with a cation-exchange resin, preferably a strongly acidic cation-exchange resin, such as SP-Sepharose FF (of the firm Pharmacia), which has been equilibrated in general in a condition of, for example, a specific conductivity of 25-34 ms/cm and a pH value of 3-4, though variable in accordance with the salt concentration, determination accuracy of pH and the species of the molecule. The above-mentioned specific conductivity value may more preferably be in the range of 30xc2x13 ms/cm and the pH value may preferably be in the range of 3.0-3.7, more preferably be 3.5xc2x10.1. While it is preferable to use a buffer solution containing dissolved therein a salt at a suitable concentration, wherein, among various possible conditions, a buffure solution of, for example, a 50-150 mM having a pH of 3-4 containing 0.25-0.32 M, preferably 0.3xc2x10.1 M NaCl may be enumerated. While there is no restriction as to the kind of the buffer solution, they may be selected among, for example, glycine-HCl, citric acid-disodium citrate, sodium citrate and acetic acid. More concretely, a 100 mM glycine-HCl buffer solution (pH=3.5) containing 300 mM NaCl may be exemplified. The specific conductivity can be determined easily using a portable conductivity meter (Model P-series CM-11P, an apparatus of To a Electronics Ltd., with standard conversion temperature of 25xc2x0 C.).
The column prepared as above is then started to pass therethrough with, for example, a 100 mM glycine-HCl buffer solution (pH=3.5, specific conductivity=31 ms/cm) containing 300 mM NaCl, whereupon an eluent fraction is obtained by collecting the passed-by eluent from the start of rising up of the 280 nm absorbance peak from the absorbance for the mere passed-by blank fraction to the end of dropping down thereof, which is then neutralized with an adequate buffer solution, whereby a high purity product of soluble TM purified up to such a degree that it has substantially no content of the substances originated from serum and from antibody can be obtained. As described afterwards, it has been confirmed that removal of the substances originated from serum and antibody can efficiently be removed with better reproducibility by using this column. Of course, they can be concentrated adequately by means of an ultrafiltration.
Further, it is also preferable to carry out exchange of the buffer solution by means of gel filtration. For example, a column of Sephacryl S-300 or S-200 which has been equilibriated with a 20 mM phosphate buffer solution (pH=7.3) containing 50 mM NaCl is loaded with a high purity product concentrated by an ultrafiltration and the column is subjected to a developing fractionation with a 20 mM phosphate buffer solution (pH=7.3) containing 50 mM NaCl, followed by collecting the fractions which show an activity for acceleration of protein C activation by thrombin when examined for such activity and performing exchange of the buffer solution, whereby a high purity product can be obtained.
In many purification processes, the aqueous injection preparation to be used according to the present invention should be obtained without necessitating any special procedure of, such as, addition and adjustment, when a solution containing the buffer component(s) and surfactant(s) permitting to reach finally the condition of pH etc. corresponding to those prescribed according to the present invention can be employed. In general, however, a practice of addition of necessary components etc. to the resulting soluble thrombomodulin solution is simple and convenient and is preferred.
For the buffer component(s) to be employed according to the present invention, there is no special restriction and any one which permits adjustment of pH at a value in the range from 5 to 7.0 and which reveals a buffering action within this range may be employed. Further, a preferred range of pH resides in 5.5-6.5. For example, one or more kinds selected from the group consisting of phosphoric acid, carboxylic acids and/or the water-soluble salts of them may be employed each in an effective amount. As the carboxylic acids and/or water-soluble salts thereof, there may be enumerated, for example, one or more selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, malonic acid, succinic acid, glutaric acid, tartaric acid, fumaric acid, malic acid and/or water-soluble salts of them, wherein special preference is given to phosphoric acid, acetic acid and/or water-soluble salts of them. There is no special restriction as to the water-soluble salts, so long as permitted pharmaceutically, and they include, for example, sodium and potassium salts.
For the container to be employed according to the the present invention, there is no special restriction so long as the material of the container and the configuration thereof are adapted for aseptic filling. There may be exemplified glass syringe (provided with a rubber cap and rubber stopper for premitting asceptic filling), glass vial (with stopper) and glass ampule as well as those made of plastic resin. A most preferred embodiment of the present invention consists in a prefilled syringe preparation filled in a syringe container. A prefilled syringe preparation is prepared by filling an aqueous injection solution preliminarily in a syringe container and can be stored/transported in this state as such, wherein the syringe container may or may not be provided with an injection needle. When it has no injection needle, an injection needle is fitted thereto usually upon the injection therewith. It may be considered to administer the preparation via penetration through skin by applying a high pressure to the aqueous preparation itself in the syringe, without using injection needle. Here, the outlet for the injection of the aqueous preparation at the front end may favorably be configured into a narrow orifice. For a means for increasing the internal pressure, there may be employed, for example, a pressing force by a compressed gas (nitrogen, helium, carbon dioxide etc.) and by a spring.
In selecting the buffer component(s) revealing a buffering action in the pH range mentioned above, such a buffer component(s) and an amount thereof may preferably be selected that the pH value of the charged preparation will not be altered beyond an extent of +0.3 before and after the container assigned for being filled is filled with the buffer component(s) to be examined and the so-filled container is subjected to a heating treatment at 50xc2x0 C. for 96 hours, as prescribed in Examination Method 1 described afterwards, though the condition may be influenced by the kind of the buffer component, its concentration and the each specific vessel to be filled. For typical buffer components as exemplified above, the concentration therefor to be employed may be in the range of, in general, at least 0.01 mM, preferably at least 1 mM, up to, in general, at the highest 1,000 mM, preferably at the highest 200 mM, especially preferably at the highest 25 mM or at the highest 20 mM. It is preferable that the glass container is preliminarily subjected to a sulfur treatment. The glass syringe may be treated by silicone coating over its inner surface.
In an embodiment of preparation of the buffer solution based on phosphate for the aqueous injection preparation according to the present invention, an aqueous solution of sodium dihydrogenphosphate (NaH2PO4 or its 12 hydrate) of a predetermined concentration and an aqueous solution of disodium hydrogenphosphate (Na2HPO4 or its dihydrate) of also a predetermined concentration were mixed together each in a definite amount to adjust the pH at the contemplated value. Alternatively, it is also possible to add an aqueous solution of sodium hydroxide dropwise to an aqueous solution of sodium dihydrogenphosphate of a definite concentration to adjust the pH of the resulting solution at the contemplated value. It is also possible to add diluted hydrochloric acid or diluted phosphoric acid dropwise to effect a minute adjustment of the pH value. In the case of a carboxylic acid salt buffer solution typically represented by an acetate buffer, an aqueous solution of acetic acid of a predetermined concentration and an aqueous solution of sodium acetate of also a predetermined concentration are mixed together each in a definite amount to adjust the pH at the contemplated value. Alternatively, it is also possible to add an aqueous solution of sodium hydroxide of a definite concentration dropwise to an aqueous solution of acetic acid of a definite concentration to adjust the pH at the contemplated value. It is also possible to employ dropwise addition of diluted acetic acid to effect a minute adjustment of the pH value.
The aqueous injection preparation according to the present invention has usually a pH value in the range from 5.0 to 7.0, preferably from 5.5 to 6.5. Especially preferably, it has a pH of about 6.0. A preferable pH range resides in, for example, 5.5-6.0.
When the concentration of a phosphate buffer solution was, varied within a wide range from 0.2 to 200 mM, a large influence was observed on a reduction in the activity of the aqueous thrombomodulin preparation at a pH of 7.3, wherein the higher the concentration of the buffer solution was, the greater was the reduction in the activity detected. For example, the residual activity of the preparation is reduced more greater at a concentration of 200 mM. For the phosphate buffer solution, however, a favorable quality with respect to the heat stability (the long term stability) of the aqueous thrombomodulin preparation can be attained when a rigorous control of pH value within the range of 5-7.0 is effected. It is preferable to adjust a pH at 5.5-6.5, in particular at about 6.0. When an adjustment of pH at a value as close to 7.0 as possible was realized, an influence on the heat stability of the aqueous thrombomodulin preparation may be caused even by a minute fluctuation in pH value and, hence, such a pH adjustment should preferably be avoided.
In an acetate buffer solution, no influence of the concentration of the buffer solution on the quality of the thrombomodulin preparation is found even when the concentration of the buffer solution is varied within a wide range from 0.2 to 200 mM. In a buffer solution of a combination of phosphate and acetate, favorable quality with respect to the heat stability is attained in a pH range from 5.0 to 7.0.
In a buffer solution of carboxylic acid salt other than acetate, a heat stability comparable to that of acetate buffer solution is realized in the same concentration range and in the same pH range as those of the acetate buffer solution. When an acetate buffer component and/or a carboxylic acid salt buffer component is added to a phosphate buffer solution, a comparable heat stability is attained.
As the surfactant to be incorporated according to the present invention, non-ionic surfactant is preferred, for example, polysorbates, such as polysorbate 80 (of the trade name xe2x80x9cTween 80xe2x80x9d) and polysorbate 20 (of the trade name xe2x80x9cTween 20xe2x80x9d); polyoxyethylene hydrogenated castor oils, such as polyoxyethylene hydrogenated castor oil 60 (of the trade name xe2x80x9cHCO-60xe2x80x9d and of the trade name of Cremophor RH 60) and polyethylene hydrogenated castor oil 50 (of the trade names xe2x80x9cHCO-50xe2x80x9d and xe2x80x9cCremophor RH 50xe2x80x9d), polyoxyethylene castor oils (of trade names xe2x80x9cCO-60TXxe2x80x9d, xe2x80x9cCO-50TXxe2x80x9d and xe2x80x9cCremophor ELxe2x80x9d etc.), ethylene oxide/propylene oxide polymers, such as polyoxyethylene(160)-polyoxypropylene(30)glycol (of the trade name xe2x80x9cPluronic F68xe2x80x9d etc.) and sorbitan sesquioleate. It is possible to use at least one of the surfactants selected from the group given above with permission of use of them in combination.
As to the shaking-stability, a turbidification preventive effect can be attained by an addition of at least 0.01% by weight for the polysorbate 80 (of trade name xe2x80x9cTween 80xe2x80x9d) and of at least 0.1% by weight for the polyoxyethylene hydrogenated castor oil (of trade name HCO-60) and the polyoxyethylene(160)polyoxy-propylene(30)glycol (of trade name xe2x80x9cPluronic F68xe2x80x9d). The polysorbate 80 (trade name xe2x80x9cTween 80xe2x80x9d) reveals a turbidification preventive effect only at an addition of 0.01% by weight and is especially preferred.
For the concentration of the surfactant to be employed according to the present invention, there may be exemplified usually a concentration of favorably at least 0.001% by weight, preferably at least 0.01% by weight, but not more than 1% by weight, preferably not more than 0.1% by weight.
The aqueous thrombomodulin preparation according to the present invention may contain, in addition to the constituent components given above, further additives including isotonicating agent (such as sodium chloride etc.) and preservative (such as p-oxybezoic acid esters etc.) as third components.
There is no special restriction as to the practical way of incorporation of the additive ingredients in the aqueous thrombomodulin preparation, as indicated above, and such a procedure may be employed, in which they are added directly to the aqueous solution containing the thrombomodulin or they are preliminarily dissolved in water, in the water for injection or in an adequate buffer solution and the resulting solution is added to the solution of thrombomodulin.
In a preferred embodiment of preparing the aqueous thrombomodulin preparation, for example, a syringe, ampule or vial is filled with a solution of thrombomodulin in water, in water for injection or in a suitable buffer solution at a concentration of 0.05 mg or more, preferably 0.1 mg or more, especially preferably 1 mg or more, per 1 ml. While the upper limit of the thrombomodulin content is not specifically restricted, a concentration of thrombomodulin of, for example, not more than 15 mg, preferably not more than 10 mg, especially preferably not more than 6 mg, per 1 ml may be exemplified. An amount of, for example, 0.3-10 ml of an aqueous solution containing the thrombomodulin at the concentration as above and other additive ingredients is filled in the container mentioned above aseptically by a conventional technique, whereby a pharmaceutical product of the aqueous injection preparation of thrombomodulin can be obtained. For sterilizing the container, ordinary practice, for example, dry heating, autoclaving and xcex3-ray sterilization, may be employed. For example, a condition of at higher than 250xc2x0 C. for more than 30 minutes for dry heating, at higher than 121xc2x0 C. for more than 20 minutes for autoclaving and irradiation of xcex3-ray at a dosage of 20-60 kGy (kilograys) may be employed. For the sterilization, dry heating is usually employed for ampule and vial made of glass and autoclaving is usually employed for the rubber stopper of vial. For sterilizing syringe, usually xcex3-ray sterilization is employed, though autoclaving may also be possible. The aqueous solution of thrombomodulin to be filled in the container is, in general, preferably caused to pass through a sterilizing filter of pore size of 0.22 or 0.2 micrometer. The aqueous injection preparation of thrombomodulin according to the present invention is prepared by aseptically filling a sterilized container with the aseptically filtered aqueous solution of thrombomodulin. It is also preferable that the so-obtained aqueous injection preparation of thrombomodulin is packaged by a sheet or in a carton.
The aqueous injection preparation of trombomodulin according to the present invention may preferably be present as a prefilled syringe preparation, as indicated above, though it may be stored in ampule or vial. In the case of ampule preparation, it is preferable to use an ampule which has been subjected to a sulfur treatment. A sulfur-treated ampule is obtained by contacting the ampule vessel with SO2 gas or favorably by spraying an aqueous ammonium sulfate solution onto the inner wall of ampule container and subjecting the so-sprayed container to a heating treatment. Usually a method of spraying 1-10% ammonium sulfate solution with subsequent heating treatment at a temperature of 630-700 Dc may be exemplified. In general, the so-treated ampule container is subjected beforehand to a further treatment on, for example, an ampule washing machine or the like, by an ultrasonication in wet state with subsequent water wash, followed by a dry heat sterilization at a temperature of 300-350 DC for several minutes, before being filled with the injection preparation.
Now, the description is directed to the method of second embodiment form of the present invention, namely, the method, wherein the aqueous injection preparation of thrombomodulin, which is characterized in that it is prepared as an asqueous solution having a pH value in the range from 5 to 7.0 and containing a soluble thrombomodulin in an effective amount and a buffer component revealing a buffering function in a pH range between 5 and 7.0 and consists of a prefilled syringe preparation filled asceptically in a syringe vessel so as to exclude any substantial gas space therein, is stored/transported in a liquid form over a long period of time.
The definitions of the soluble thrombomodulin, the buffer component and the aqueous injection preparation of thrombomodulin are the same as described previously.
For the syringe vessel to be used according to the present invention, use of commercial injection syringe may be preferred and, in general, a syringe vessel for prefilled syringe having an inner diameter of about 8.6 mm, about 6.3 mm or about 4.6 mm may be exemplified. Though selected in accordance with the charge amount of the aqueous solution of thrombomodulin, one having an inner diameter of about 4.6 mm is at the most preferable in respect of the shaking-stability, though the syringe vessel of about 6.3 mm inner diameter permits enough application. For syringe vessel of inner diameter of about 8.6 mm, it is necessary to keep the proportion of gas space in the vessel when charged with the aqueous preparation at the most 50 volume %. Thus, the syringe vessel of inner diameter of about 8.6 mm can ensure its shaking-stability so long as the proportion of gas space therein after having been filled with the aqueous preparation is kept at the most 50 volume %.
The aqueous injection preparation of trombomodulin may not, according to the specific shape and inner size of the vessel, substantially be subjected to shaking due to the surface tension thereof. In the context of this specification, xe2x80x9cportion subject to shakingxe2x80x9d does meen a portion of the vessel at which the aqueous injection preparation of thrombomodulin present in the vessel can be brought into motion of substantial shaking when the vessel is shaken under a condition of an amplitude of 5 cm and a frequency of 180 turns per minute at a temperature of 25xc2x0 C. xe2x80x9cThe proportion of gas spacexe2x80x9d in the vessel is a value calculated by dividing the volume difference calculated by subtracting the volume of the aqueous injection preparation of thrombomodulin present within the portion subject to shaking from the volume of the portion subject to shaking, by the volume of the portion subject to shaking of the vessel, expressed in percent value. The amount of aqueous injection preparation of thrombomodulin present in the portion subject to shaking, namely, the movable amount of the aqueous injection preparation in the vessel can be confirmed by, for example, repeating an experiment for ascertaining and measuring the amount of the aqueous preparation in the vessel, which has moved downwards in the vessel after keeping the vessel filled with the aqueous injection preparation of thrombomodulin for 5 seconds in such a state, that the vertical axis and the horizontal axes of the vessel are held fixed, after it has been shaken for two minutes under the condition given above. Hereby at the same time, it is also possible to confirm the portion subject to shaking, so that the volume thereof can be determined. In the case of a syringe of simple form, the portion subject to shaking consists, of course, of the space confined between the syringe and the stopper (and the cap) itself. In the case of the syringe of about 4.6 mm inner diameter, no substantial shaking occurs and it is unnecessary to take into account of the proportion of gas space.
Usually, the syringe vessel which is sealed by a cap is filled with the aqueous injection preparation of thrombomodulin on an ordinary manner and is then fitted with a stopper. For fitting the stopper into the syringe vessel, a technique of, for example, vacuum fitting and use of a vent tube (or a sleeve) may be employed. When the syringe vessel is sealed by vacuum fitting of stopper, the proportion of gas space can relatively easily be adjusted at a value of, for example, not higher than 15 volume %. Therefore, it is to be understood by xe2x80x9cexclude any substantial gas spacexe2x80x9d prescribed according to the present invention as a preferred embodiment that typically the proportion of gas space is not higher than 15 volume %. It is also preferred that the proportion of gas space is not higher than 10 volume %, much more that it is not higher than 5 volume %. With a syringe vessel for a prefilled syringe adapted for subcutaneous injection or for intramuscular injection (having ordinarily a capacity of several ml, preferably not greater than 2 ml, more preferably not greater than 1 ml, especially preferably not greater than 0.5 ml) having an inner diameter of at least about 8.6 mm, a prefilled syringe preparation exhibiting a superior shaking-stability can easily be attained, so long as the proportion of gas space remains below the above-given value.
Beside the syringe vessel explained above, vials and ampules may also be employed for the vessel when taking into account of incorporation of a surfactant. Ordinary vials and ampules have relatively large inner diameter and, hence, it is relatively difficult for them to decrease and adjust the proportion of gas space. Therefore, use of vials and ampules may preferably be avoided, when the proportion of gas space should be adjusted precisely.
The aqueous injection preparation of thrombomodulin prepared as above exhibits a long term stability and, thus, it can afford to preserve at least about 80% of its original activity over a period of 12 months, preferably 18 months, at 5xc2x0 C. In some cases, it may permit storage/transportation over a long period of time even exceeding two years, preferably up to three years, so long as certain other conditions are satisfied.
The aqueous injection preparation of thrombomodulin according to the present invention will not suffer from occurrence of turbidity even under a condition of shaking in an amplitude of 5 cm, at a frequency of 180 turns per minute at 25xc2x0 C. for one month. Thus, the aqueous injection preparation of thrombomodulin according to the present invention exhibiting such sufficient stabilities as given above can be stored/transported in a form of liquid over a long period of time.
The condition of storage/transportation to be encountered by the aqueous injection preparation of thrombomodulin according to the present invention may include a temperature higher than that causing it to freeze but not higher than room temperature and, when exemplified concretely, from 0xc2x0 C. to 20xc2x0 C., preferably at around 5xc2x0 C. The permissible duration therefor may cover, in general, 12 months, preferably 18 months, more preferably about two years.
The aqueous injection preparation of thrombomodulin according to the present invention provides for administration forms of subcutaneous and intramuscular injections. Thus, the fourth aspect of the present invention consists in a method for applying the prefilled syringe preparation of thrombomodulin to adminitration to the patient via subcutaneous or intramuscular injection.
As the fifth aspect of the present invention consists in a method for maintaining the concentration of a soluble thrombomodulin in blood, characterized in that an aqueous injection preparation of thrombomodulin for sustained effectiveness containing an effective amount of the soluble thrombomodulin is administered to the patient via subcutaneous or intramuscular injection at an adminitration frequency of once per 2 to 5 days.
The sixth aspect of the present invention resides in a durable aqueous injection preparation of thrombomodulin containing, as the active ingredient, a soluble thrombomodulin to be administered to the patient by subcutaneous or intramuscular injection at an adminitration frequency of once per 2 to 5 days.
Since thrombomodulin exists on the surface of vascular endothelial cells and the site of action thereof is limited to within blood vessel, it has been accepted that administration of thrombomodulin by intravenous injection may bring about a direct effect and is most preferable. For example, Japanese Patent Kokai Sho 64-6219 discloses an example of drip intravenous injection thereof.
However, it is necessary to develop a medicinal preparation and so on which have conventionally not been found and which may permit an adequate choice in accordance with the condition of patient and with the practical convenience.
In the reports and publications so far, it has not always been made clear that thrombomodulin is administered to a patient intentionally via a route other than intravenous injection. Therefore, the inventors of the present invention had made research therefor and found, for the first time, that a concentration of thrombomodulin in blood can be built up, when the soluble thrombomodulin constituted of the amino acid sequence composed of amino acid residues from the site No. 19 to the site No. 516 of sequence listing SEQ ID NO: 1 (a soluble thrombomodulin obtained by transfecting the DNA segment which codes the amino acid sequence of the sequence listing SEQ ID NO: 1 to a host cell) is administered to a patient by subcutaneous injection, and that, in particular, this thrombomodulin concentration in blood is preserved over a remarkably longer time as compared with the soluble thrombomodulin constituted of the amino acid sequence composed of the residues from the site No. 367 to the site No. 480 of the sequence listing SEQ ID NO: 1.
Therefore, a preferred sustained medicinal preparation of thrombomodulin is provided according to the present invention.
It may also be favorable that a local anesthetic is further incorporated in the durable medicinal preparation mentioned above. A further content of a preservative is also preferred.
Preferred examples of the local anesthetic agent include procaine hydrochloride and benzyl alcohol. The amount of administration of the local anesthetic agent may usually be, for example, in the range of 0.5-10%, preferably 1-5%, based on the total weight of the injection preparation.
It is permissble, if necessary, that the injection preparation contains amino acids, salts, carbohydrates, surfactants, albumin, gelatin and so on, as disclosed in Japanese Patent Kokais Sho 64-6219 A and Hei 6-321805 A and so on. Addition of preservative is also favrable, wherefore preferred examples include paraoxybenzoic acid esters, such as methyl paraoxy-benzoate, ethyl paraoxybenzoate and mixtures of paraoxybenzoic acid esters. The amount of the preservative may usually be, for example, in the range from 0.01 to 1.0%, preferably from 0.1 to 0.3%, based on the weight of the injection preparation.
For the soluble thrombomodulin to be employed in the sustained medicinal preparation according to the present invention, every soluble thrombomodulin can be incorporated without any special restriction, wherein preference is given, in particular, to a soluble thrombomodulin constituted of the amino acid sequence composed of the amino acid residues from the site No. 19 to the site No. 516 of the sequence listing SEQ ID NO: 1, a soluble thrombomodulin constituted of the amino acid sequence composed of the amino acid residues from the site No. 19 to the site No. 516 of the sequence listing SEQ ID NO: 2, a soluble thrombomodulin obtained by transfecting the DNA segment which codes the amino acid sequence of the sequence listing SEQ ID NO: 1 to a host cell and a soluble thrombomodulin obtained by transfecting the DNA segment which codes the amino acid sequence of the sequence listing SEQ ID NO: 2 to a host cell.
The sustainability of the medicinal preparation according to the present inevention may favorably be exemplified by the fact that the half life of the concentration in blood (T1/2) is at least 16 hours and that the mean retention time (MRT) in plasma is at least 36 hours.
It is preferred that the sustainable medicinal preparation according to the present invention is provided in a form of aqueous injection preparation. While the sustained medicinal preparation according to the present invention may be distributed in a form of freeze-dried product which is to be dissolved in water upon its administration, it is most convenient to use a prefilled syringe preparation prepared by filling a syringe vessel with the aqueous injection preparation of thrombomodulin described above aseptically as a subcutaneous or intramuscular injection medicine as such.
The manner of practical procedure for incorporating the additives in preparing the durable preparation according to the present invention is not specifically restricted. Thus, for example, the additives are admixed to an aqueous solution of thrombomodulin or are preliminarily dissolved in water, in water for injection or in an adequate buffer solution and the resulting solutions are then brought together in a suitable proportion before preparing the aqueous injection preparation. For example, an aqueous solution containing 0.05-15 mg, preferably 0.1-6 mg, of the soluble thrombomodulin together with the above-mentioned additives per 1 ml of water, water for injection or an adequate buffer solution is charged in, for example, a syringe vessel, a vial or, occasionally, an ampule in an amount of, for example, 0.5-10 ml, and the thereby formulated preparation is served as such for the aqueous injection preparation of thrombomodilin or, in the case of vial and ampule, the formulated preparation may further be subjected to freeze-drying. Usually the so-obtained durable medicinal preparation may contain a soluble thrombomodulin in an amount of, for example, 0.01 to 100 mg.
The administration frequency of the aqueous injection preparation of thrombomodulin according to the present invention may be, as in conventional practice, 1 to 3 administrations per day, while it is possible to select an administration frequency of, for example, once per 2-5 days. Especially, in the case of subcutaneous or intramuscular injection, an administration frequency of, for example, once per 2-5 days is preferred due to its durability. For the dosage per one administration, while it is permissible to administer the maximum permissible dose by a drip intravenous injection, usually a dosage for one single administration of at the most 1 mg of the soluble thrombomodulin per kg of the body weight is exemplified for a one-shot administration into blood vessel. For the minimum dosage, usually an amount of at least 0.001 mg, preferably at least 0.005 mg per 1 kg of the body weight may be exemplified. The upper limit of dosage for one single subcutaneous or intramuscular injection is determined by the maximum soluble amount of the active ingredient in one injection amount of the carrier liquid. A typical dosage adapted for subcutaneous or intramuscular injection is, in general, several milliliters, preferably at the most 2 ml, more preferably at the most 1 ml, especially preferably at the most 0.5 ml. Thus, a dosage expressed by the weight of the soluble thrombomodulin for one single administration by subcutaneous or intramuscular injection may usually be, for example, 20 mg or less, wherein the minimum effective amount for one single administration may, for example, be at least 0.001 mg per 1 kg of the body weight, preferably at least 0.005 mg per 1 kg of the body weight, as indicated above.
The durable preparation of thrombomodulin according to the present invention can reveal its durability by administration not only via subcutaneous but also via intramuscular route, wherein preference is given to subcutaneous injection.
It is possible according to the present invention to remarkably extend the duration in which the concentration of thrombomodulin in blood is maintained, which results in a possible reduction of administration frequency, whereby a medicinal preparation of soluble thrombomodulin effective with smaller amount as compared with that of conventional preparation for intravenous injection is provided. Such medicinal preparation can reduce patient""s pain upon the injection and may permit occasionally injection by the patient himself, contributing thus to patient""s therapeutic convenience considerably.
The examination for the acute toxicity of the aqueous injection preparation of soluble thrombomodulin according to the present invention has revealed that no mortal instance was found upon an administration to groups of each five male+female SD rats by intravenous injection at a dose of 180 mg/kg as the dose weight of the thrombomodulin. Also no mortal instance was seen upon administration by subcutaneous injection at the same dose of 180 mg/kg.